Methods that allow accurate detection and quantitation of nucleic acid sequences are invaluable tools for diagnosis and treatment of a wide range of diseases. For example, detection and quantitation of an infectious agent's nucleic acid sequence can provide diagnostic or prognostic information or allow a physician to monitor a patient's response to therapy. Further, accurate detection of low levels of viremia in infections such as human immunodeficiency virus (HIV) or hepatitis C virus helps to prevent spread of the virus, to estimate prognosis and response to therapy, and to detect the emergence of drug-resistant viruses in treated individuals. It is equally important to accurately measure high levels of viremia in untreated patients to establish initial infection levels.
Methods for amplifying a target nucleic acid sequence that may be present in a test sample are known and include methods such as the polymerase chain reaction (PCR; e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,965,188, 6,040,166, 6,197,563 and 6,514,736); reverse transcription polymerase chain reaction (RT-PCR; e.g., U.S. Pat. Nos. 5,310,652 and 5,322,770); transcription-mediated amplification (TMA; e.g., U.S. Pat. Nos. 5,399,491, 5,824,518 and 7,374,885); ligase chain reaction (LCR; e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663); strand displacement amplification (SDA; e.g., U.S. Pat. Nos. 5,422,252, 5,547,861 and 5,648,211); rolling circle amplification (RCA; e.g., U.S. Pat. No. 5,648,245 and U.S. Pat. No. 5,854,033); helicase-dependent amplification (HDA; e.g., U.S. Pat. Nos. 7,282,328 and 7,662,594); and nucleic acid sequence based amplification (NASBA; e.g., U.S. Pat. No. 5,130,238).
Methods for detecting non-amplified or amplified target nucleic acid sequences that may be present in a test sample are also known. Some detection methods are “homogeneous” methods that do not require separation of a detection agent associated with the target nucleic acid from the detection agent that is not associated with the target nucleic acid. Such methods include use of intercalating dyes (e.g., U.S. Pat. Nos. 5,312,921, 5,814,447, 6,063,572, 6,541,205 and 6,569,627), a Hybridization Protection Assay (HPA; e.g., U.S. Pat. No. 5,283,174), and use of molecular beacon probes (e.g., U.S. Pat. No. 5,925,517) or molecular torch probes (e.g., U.S. Pat. No. 6,361,945). Other methods are “heterogeneous” and require physical separation of the detection agent associated with the target nucleic acid from the detection agent not associated with the target nucleic acid. One of the most common heterogeneous methods involves the capture of target nucleic acids onto a solid support, hybridization of a labeled detection probe, and washing under appropriately stringent conditions to remove non-specifically bound probe. The label (e.g, a radioisotope) that remains bound to the support is then measured.
While currently available amplification techniques may provide sufficient sensitivity for some applications, other diagnostic and therapeutic situations require more sensitivity than is available from these available methods. Furthermore, under certain circumstances, it is desirable to determine whether the nucleic acid sequence is present at a high or low concentration level. Thus, there is a need for methods through which target nucleic acids in a test sample can be detected and quantified accurately over a relatively large dynamic range in a single experiment.
Nucleic acid analytes of interest from a specimen may be present at concentrations that are less than can be detected by routine methods. This problem is often circumvented by implementing one of several nucleic acid amplification techniques (vide supra) prior to detection. The nucleic acid analytes of interest from a specimen may also be present in very small numbers (e.g., 1, 7, 29, etc., or zero in the case of a non-infected specimen) or larger numbers ranging into the millions, billions or more. Therefore, it is critical for quantification that the nucleic acid analytes are amplified proportionally to accurately reflect their initial numbers. Attempts to achieve reproducible quantitation of nucleic acids over a dynamic range by using nucleic acid amplification methods has been a challenging endeavor and a number of problems have been encountered including, for example, the need for amplification internal standards and (even very slightly) different amplification efficiencies of internal standards (Clin. Chem. 40(4): 630-636 (1994); Clin. Chem. 41(8): 1065-1067 (1995); Biochim. Biophys. Acta 1219(2): 493-498 (1994); Biotechniques 15(1): 134-139 (1993); J. Infect. Dis. 165(6): 1119-1123 (1992); Proc. Nat. Acad. Sci. USA 86(24): 9717-9721 (1989)). Two basic approaches have been used to solve these problems, with mixed success.
In the first basic approach, the concentration of a target nucleic acid sequence present in a test sample is determined based on the rate at which amplicons are produced over time. An example of this approach is known as “real-time PCR;” increasing amplicon levels are measured with probes that change their fluorescent properties in response to increasing amplicon concentration. The time it takes for the amplicon to reach a predefined level is correlated with the concentration of the target nucleic acid sequence present in the sample by comparing the results of the experimentally detected amplicon to results from standards that contain a range of known amounts of the target nucleic acid sequences. Multiple standards may be used, often ranging from the low to high ends of expected responses from the analyte of unknown concentration. In amplification systems in which the reaction is divided into cycles, such as the PCR or LCR, precision is limited by the amount of amplification that occurs within each cycle; for the PCR, the amount is theoretically a two-fold increase per cycle. However, for amplification systems that lack discrete amplification cycles, such as TMA or SDA, rate changes may be difficult to measure accurately because amplicon concentration can change rapidly over a very short period of time. The accuracy of time and amplicon concentration measurements greatly affects the ability to correlate measured results with an initial target nucleic acid concentration with any degree of precision.
In the second basic approach, the amplification reaction is run for a fixed time, and the amount of amplicon produced is measured and correlated to the concentration of target nucleic acid sequence present in the test sample. Such methods are sometimes referred to as “endpoint” methods because the amplicons are measured at a single point at the end of the reaction rather than at multiple time points during the amplification reaction. These methods typically require the use of an excess of nucleic acid probe relative to the target nucleic acid so that the amount of probe hybridized relates directly to the amount of target present and avoids signal saturation due to excess target, referred to as “target saturation” (e.g., U.S. Pat. No. 4,851,330). If target saturation occurs, the probe signal reaches a plateau and the upper end of the dynamic range is truncated. When large amounts of amplicon are produced, even larger amounts of probe must be used to extend the dynamic range, and the amplicon-probe hybridization signal may exceed the detector's capacity for accurate detection. Furthermore, the background signal is often elevated as probe concentration increases, which limits sensitivity of detection at the lower end of the dynamic range. Lowering the specific activity of the probe can be used to reduce the background signal; however, the sensitivity of detection at low target levels is also often decreased. Thus, optimization of endpoint detection may reduce accuracy on one or both ends of the dynamic range.
Generally, while known amplification systems have been optimized to produce amplicons quantitatively over a large dynamic range, detection systems have been unable to perform accurately over that same broad range. This limitation requires additional steps in the process, such as serial dilution of the amplicons or physical separation steps, in order to reduce non-specific background signal and to measure the amplicon concentration across the full dynamic range. Physical separation or dilution steps increase the complexity and length of the test procedure. Manipulating amplicon products increases the danger of cross-contaminating samples or amplification reaction mixtures, which could lead to false positive results. In addition, amplicon manipulation increases the possibility of errors in quantification, including false negative results. Ideally, a nucleic acid detection and quantitation system should include an amplification reaction that generates measurable amounts of one or more amplicons reproducibly and proportionately across a wide dynamic range, as well as a detection system that can quantitate the amplicons across the same dynamic range.
In certain known methods, a nucleic acid hybridization reaction is used to determine the amount of amplicon by correlating either the rate of hybridization or the extent of hybridization to the amount of amplicon present. However, kinetic measurements of hybridization pose many of the same disadvantages of kinetic measurements of amplification reactions and are thus not preferred.
In some variations of known in vitro nucleic acid amplification procedures, researchers have sought to achieve an extended dynamic range by individually adjusting the efficiencies of amplification of one or more target sequences by optimizing the reaction conditions for each target nucleic acid. However, such optimization generally requires complex and extensive experimentation and may not yield reproducible results because of slight differences between samples, presence of inhibitors in reaction mixtures, variations in test reagents and/or their quantities in individual reaction mixtures, or conditions in which the reactions are performed. Thus, there remains a need to extend the dynamic range of quantitative endpoint assays that does not require excess experimentation and provides a robust system with wide dynamic range.
Compositions and methods that respond to this need would allow quantitative measurement of a desired target nucleic acid in a sample by using nucleic acid amplification and detection of the amplified products over an extended dynamic range. The compositions and methods described herein provide a simple solution to problems associated with previous quantitative nucleic acid amplification and detection methods.